SALMONID DISEASE INVESTIGATIONS

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COLORADO DIVISION OF WILDLIFE
WILDLIFE COMMISSION
Tom Burke, Chair Claire O’Neal, Vice Chair Robert Bray, Secretary Dennis Buechler Brad Coors Jeffrey Crawford Tim Glenn Roy McAnally Richard Ray Ex Officio Members Harris Sherman John Stulp, Dept. of Ag.
AQUATIC RESEARCH STAFF
Mark S. Jones, General Professional VI, Aquatic Research Leader Arturo Avalos, Hatchery Technician III, Research Hatchery Stephen Brinkman, General Professional IV, F-243, Water Pollution Studies Harry Crockett, General Professional IV, Eastern Plains Native Fishes Patrick Martinez, General Professional V, F-242, Coldwater Reservoir Ecology & GOCO-Westslope Warmwater R. Barry Nehring, General Professional V, F-237, Stream Fisheries Investigations Kevin B. Rogers, General Professional IV, GOCO-Colorado Cutthroat Studies Phil J. Schler, Hatchery Technician V, Research Hatchery George J. Schisler, General Professional IV, F-394, Salmonid Disease Studies Kevin Thompson, General Professional IV, F-427, Whirling Disease Habitat Interactions, GOCO-Boreal Toad Studies Harry Vermillion, Scientific Programmer/Analyst, F-239, Aquatic Data Analysis Nicole Vieira, Physical Scientist III, Water Quality Studies Rosemary Black, Program Assistant I Paula Nichols, Federal Aid Coordinator
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Prepared by: George J. Schisler, GP IV, Aquatic Researcher Approved by: ____ _____________________________________ Mark S. Jones, Aquatic Wildlife Research Leader Date: The results of the research investigations contained in this report represent work of the authors and may or may not have been implemented as Division of Wildlife policy by the Director or the Wildlife Commission
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Signature Page ……………………………………………………………………..ii List of Tables ………………………………………………………………………v List of Figures …………………………………………………………………......vi
Job No. 1. Breeding and Maintenance of Whirling Disease Resistant Rainbow Trout Stocks. Hatchery Production……………………………………………………...... 1 Research Projects…………………………………………………………... 3 Job No. 2. Whirling Disease Resistance Laboratory Experiments. Experiment 1: Inheritance of Myxobolus cerebralis resistance among second generation crosses of the Hofer (GR) and Colorado River (CRR) rainbow trout strains. Introduction………………………………………………………………… 4 Methods……………………………………………………………………..4 Results……………………………………………………………………… 8 Discussion………………………………………………………………….. 8 References…………………………………………………………………..12 Experiment 2: Physiological characteristics and inheritance of Myxobolus cerebralis resistance among multiple generational crosses of the Hofer (GR) and Colorado River (CRR) rainbow trout strains.
Introduction…………………………………………………………………13
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Field Performance Evaluations: Comparison of Hofer (GR) and Tasmanian strain rainbow trout as catchable plants in two put-and-take waters in Colorado. Introduction………………………………………………………………… 41 Methods……………………………………………………………………..41 Results……………………………………………………………………… 43 Discussion………………………………………………………………….. 48 References………………………………………………………………….. 49 Job No. 4. Whirling Disease Resistant Wild Strain Brood Stock Development and Evaluation. Introduction………………………………………………………………… 50 Methods……………………………………………………………………..51 Results……………………………………………………………………… 53 Discussion………………………………………………………………….. 59 Job 5. Technical Assistance Technical Assistance Milestones…………………………………………... 60 Appendix I. Creel Survey Reports for Flatiron and Pinewood Reservoirs Flatiron Reservoir (Water Code 54851)…………………………………… 63 Pinewood Reservoir (Water Code 55928)…………………………………. 77 Appendix II. Molecular Techniques for Identifying Hofer (GR) Strain Rainbow Trout Colorado River Rainbow (CRR) vs. Hofer (GR) AFLPs Progress Report-
Pisces Molecular ………………………………………………………….102
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TABLES Table 1.1. Fish Research Hatchery on-site spawning information for Hofer
(GR) and Harrison Lake rainbow trout strains during the winter 2006-2007 spawning season….. ..................................................................3
Table 2.1. Family groups created for M. cerebralis resistance laboratory testing
(Fish Research Hatchery (FRH) brood) during spring 2006 spawning season...........................................................................................................6
Table 2.2 Family groups created for M. cerebralis resistance laboratory testing
(Colorado Cooperative Fish and Wildlife Unit wet lab (COOP) brood) during spring 2006 spawning season ...............................................7
Table 2.3. Family groups created for M. cerebralis resistance experiment
conducted at the COOP wet lab in 2006-2007...........................................16 Table 2.4. Control groups separated from family groups created for M.
cerebralis resistance experiment conducted at the COOP wet lab in 2006-2007 ..................................................................................................20
Table 2.5. Batch weights and feed amounts for families on size 1 trout diet for
M. cerebralis resistance experiment conducted at the COOP wet lab in 2006-2007 ..............................................................................................21
Table 3.1. Results of M. cerebralis infection evaluations at the Chalk Cliffs
Rearing Unit...............................................................................................37 Table 3.2. Results of M. cerebralis infection evaluations at the Poudre Rearing
Unit... .........................................................................................................38 Table 3.3. GR and Tasmanian strain rainbow trout stocked from April through
June, 2006, at Flatiron and Pinewood reservoirs. ......................................42 Table 4.1. Myxospore counts and classification of age 1+ and 2+ rainbow trout
based on fin clips and AFLP analysis in the Gunnison River, 2006..........55
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Table 4.2 Growth and myxospore counts for wild CRR and stocked B2 fish downstream of Stagecoach Reservoir and Service Creek..........................58
FIGURES Figure 2.1. Average spore counts for the three Hofer (GR), three Colorado River
Rainbow (CRR), ten F1 [GR-CRR (50:50)] and 16 B2 [GR-CRR (25:75)] strains ...........................................................................................10
Figure 2.2. Example of inter-family variability in infection severity in F1 and B2
strains .........................................................................................................11 Figure 3.1. Daily mean temperature, cumulative temperature units, and sample
collections at the Chalk Cliffs Rearing Unit from January 2005 to January 2006 ..............................................................................................35
Figure 3.2. Daily mean temperature, cumulative temperature units, and sample
collections at the Poudre Rearing Unit from July 2005 to July 2006 ........35 Figure 3.3. Growth rates for the Hofer (GR) and Tasmanian strain rainbow trout
at the Chalk Cliffs Rearing Unit ................................................................36 Figure 3.4. Myxospore counts for Hofer (GR) and Bellaire rainbow trout at four
months, eight months, and one year at the Poudre Rearing Unit...............36 Figure 3.5. Catch data from creel reports for number of rainbow trout caught by
strain at Flatiron Reservoir.........................................................................44 Figure 3.6. Catch data from creel reports for number of rainbow trout caught by
Pinewood reservoirs in 2006......................................................................46 Figure 3.9. Characteristics of fish contributing to angler preference at Flatiron
and Pinewood reservoirs in 2006...............................................................47 Figure 3.10. Angler preference for trout flesh color, Flatiron and Pinewood
State: Colorado Project No. F-394-R6 Project Title: Salmonid Disease Studies:
Whirling Disease-Resistant Rainbow Trout Studies Period Covered: July 1, 2006-June 30, 2007 Project Objective: Development of rainbow trout brood stocks resistant to M.
cerebralis for both hatchery and wild fish management applications. Job No. 1. Breeding and Maintenance of Whirling Disease Resistant Rainbow
Trout Stocks Job Objective: Rear and maintain stocks of whirling disease resistant rainbow trout
stocks. Hatchery Production
The whirling disease resistant rainbow trout brood stocks reared at the Fish Research Hatchery, Bellvue, CO (FRH) are unique and each requires physical isolation to avoid unintentional mixing of stocks. Extreme caution is used throughout the rearing process and during on-site spawning operations to ensure complete separation of these different brood stocks. All lots of fish are uniquely fin-clipped and some unique stocks are individually marked with Passive Integrated Transponder (PIT) tags before leaving the main hatchery. This allows for definitive identification before the fish are subsequently used for spawning.
Starting in the middle of October 2006, FRH personnel checked all of the Hofer0F
* (GR) and Harrison Lake brood fish (2, 3, 4 and 5 year-olds) weekly for ripeness.
Maturation is indicated by eggs or milt flowing freely with slight pressure applied
to the abdomen of the fish. The first females usually maturate two to four weeks after the first group of males. As males are identified, they are moved into a separate section of the raceway to reduce handling and fighting injuries. On November 16, 2006, the fish from the first group of GR females were ripe and ready to spawn. Before each fish was spawned, it was examined for the proper identification (fin-clip or PIT tag). This procedure was repeated each time ripe females were spawned throughout the winter.
The wet spawning method was used, where eggs from the female are stripped into a bowl along with the ovarian fluid. After collecting the eggs, milt from several males is added to the bowl. Water is poured into the bowl to activate the milt. The bowl of eggs * Hofer is used interchangeably with GR throughout this document to describe the resistant strain of rainbow trout obtained in 2003 from facilities in Germany.
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and milt is then covered and not disturbed for several minutes while the fertilization process takes place. The eggs are then rinsed with fresh water to expel old sperm, feces, egg shells and dead eggs. The eggs are then poured into an insulated cooler to water- harden for approximately one hour. The water-hardened fertilized (green eggs) from all the different crosses of the GR and Harrison Lake strains were moved to the FRH main hatchery building. Extreme caution was used to keep each individual cross totally separate from all others. Upon reaching the hatchery the green eggs are tempered and then disinfected (PVP Iodine, Western Chemical Inc., Ferndale, Washington, at 100 ppm for 10 minutes at a pH of 7). Eggs were then put into vertical incubators (Heath Tray, Mari Source, Tacoma, Washington) with 5 gpm of 12.2º C (54º F) of flow-through water. The total number of eggs was calculated using number of eggs per ounce (Von Bayer trough count minus 10%) times total ounces of eggs. Separate daily egg-takes and specific individual crosses were put into separate trays and recorded. To control fungus, eggs received a prophylactic flow-through treatment of formalin (1,667 ppm for 15 minutes) every other day until eye-up. On the 14th day in the incubator at 12.2º C (54º F), the eggs reach the eyed stage of development. The eyed eggs are removed from the trays and physically shocked to detect dead eggs, which turn white when disturbed. Dead eggs were removed (both by hand and with a Van Gaalen fish egg sorter, VMG Industries, Grand Junction, Colorado) on the 15th day. The total number of good eyed eggs was calculated using the number of eggs per ounce times total ounces. On the 16th day the eyed eggs were shipped via insulated coolers to other state agency hatcheries. The whole process was repeated throughout the spawning season with separate crosses of GR and Harrison Lake rainbow trout. The GR and Harrison Lake rainbow trout production on-site spawn started on November 16, 2006 with ripe GR females. The last group of Harrison Lake females was spawned on February 1, 2007. With a goal in the fall to produce @ 200,000 eyed eggs, the egg take far exceeded the production needs with over 442,500 eyed eggs produced (Table 1.1). With the availability of both ripe males and females of several year classes and combinations of previous years crosses (F1 and B2) of GR and Harrison Lake strains, FRH personnel produced over 20 different lots during the spawn take. Surprisingly the overall egg quality remained quite good with 1st egg pick-off of only 26%. FRH personnel were able to fill all GR egg requests for Colorado, California, and Utah for both production and research directed projects in 2006-2007.
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Table 1.1. Fish Research Hatchery on-site spawning information for GR and Harrison Lake rainbow trout strains during the winter 2006-2007 spawning season.
STRAIN (CROSSES)
DATE SPAWNED
SHIPPED TO
100% GR 11/29/06-1/11/07 101 299,250 212,400 CO and CA State Hatcheries/Research
75% GR 25% Harrison Lake
11/16/06-1/30/07 92 266,600 202,300 CO and UT State Hatcheries/Research
50% GR 50% Harrison Lake
1/04/07-1/19/07 15 21,350 16,350 CO Hatcheries
100% Harrison Lake 1/04/07-2/01/07 12 15,300 11,800 CO Hatcheries Research Projects Eggs produced specifically for research projects comprise a large proportion of the total production from the FRH. Specific details of those individual crosses and families created for the laboratory and field experiments are described in their respective sections of this report. The bulk of these family group descriptions appear in the following section, Job 2: Whirling Disease Resistance Laboratory Experiments.
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Job No. 2: Whirling Disease Resistance Laboratory Experiments Job Objective: Evaluate the inheritability and stability of whirling disease resistance in
selected strains of rainbow trout. Experiment 1: Inheritance of Myxobolus cerebralis resistance among second
generation crosses of the Hofer (GR) and Colorado River (CRR) rainbow trout strains.
INTRODUCTION
The Hofer (GR) rainbow trout strain has been identified as more resistant to
whirling disease than other rainbow trout strains when exposed to Myxobolus cerebralis in laboratory conditions (Hedrick et al. 2003). However, the survival and viability of the strain in the wild is questionable and the consequences of stocking the strain directly into wild trout waters is unknown (Schisler et al. 2006). In 2004, a study was conducted in which GR strain rainbow trout and Colorado River rainbow (CRR) strain rainbow trout were crossed. The principle aim of that project was to incorporate whirling disease resistance from the GR into the CRR strain, a strain that is typically used to establish wild rainbow trout populations in Colorado (Schisler et al. 2006). Results of exposure experiments with the GR-CRR (50:50) cross (F1 generation) showed that spore counts per fish were reduced significantly from those found in the pure CRR strain. While average infection severity in the first generation cross was much lower than the pure CRR strain, it was not reduced to the spore count levels of the pure GR strain. However, some families, created from individual male-female pairs, were more resistant than others. In addition, many individual fish from those crosses appeared to inherit a similar level of resistance as observed in the pure GR strain. A second exposure experiment was initiated to evaluate the performance of the pure GR, pure CRR, F1 generation, and a second generation GR-CRR (25:75) backcross (defined as the B2 generation) in the presence of the whirling disease parasite. This experiment would provide insight to the continued inheritability of resistance to M. cerebralis, particularly in F1 generation fish back- crossed with the wild CRR strain.
METHODS Spawning of all families occurred at the Colorado Division of Wildlife Fish Research Hatchery (FRH) and Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab from mid-November 2005 through the end of December 2006 (Tables 2.1 and 2.2). Both male and female pure GR and F1 fish are held on site. F1 individuals had been tagged with Passive Integrated Transponder (PIT) tags to identify them by family group. Only the lowest spore count families of the F1 variety were retained for this second generation of crosses. These fish were identified by their 10 digit alpha numeric code prior to spawning. All tagged or untagged individuals were also numbered in the order that they were spawned. Pure CRR individuals were held at the Colorado Division of
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Wildlife Glenwood Springs Hatchery (GWSH). Males were spawned at the GWSH and their sperm was transported in individual, numbered containers back to the FRH for fertilization of the GR and F1 eggs. In addition, live male and female CRR rainbow trout were transported back to the FRH and spawned with each other as well as GR and F1 males. An anal fin clip was taken from each spawned individual and stored in 70% ETOH for later genetic analysis. Eggs were placed in incubators at the FRH or COOP wet lab and held until they were eyed. Once eyed, eggs were placed in 76 liter (20 gallon) tanks containing short (7 cm) standpipes for a greater amount of water turnover at the COOP wet lab, where they were hatched.
Individual families (single male/female matings) were used as replicates in this experiment. Three pure GR families, three pure CRR families, 10 F1 families, and 16 B2 families were used in this evaluation. In some cases, up to 2,000 fertilized eggs are produced with each paired cross. For the purposes of this exposure experiment, fish were culled down to approximately 50 per family until immediately before exposure. At that time the families were then reduced to 30 fish each.
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Table 2.1. Family groups created for M. cerebralis resistance laboratory testing (FRH brood) during spring 2006 spawning season.
G ro up F em ale M a le F ert ilized F em ale O rig in M ale O rig in H atc hed D o s ed Lab E xperim ent O ther D es t ina t io ns 1 135113115a 134959197a 11/22/2005 H o ferxH arris o n (54) (04 EX P ) H o fe r (56 ) (04 EX P ) 12/19/2005 2 /17 /2005 E9 Utah 12,992 eggs 12-13-05 2 133754663a 134839090a 11/22/2005 H o fer (13 ) (04 EX P ) H o fe rxH arris o n (48) (04 E X P ) 12/19/2005 2 /17 /2005 E8 " " 3 133754663a 134661691a 11/22/2005 H o fer (13 ) (04 EX P ) H o fe r (15) (04 E X P ) 12/19/2005 2 /17 /2005 E7 " " 4 133957366a 134609145a 11/22/2005 H o fer (14 ) (04 EX P ) H o fe rxH arris o n (53) (04 E X P ) 12/19/2005 2 /17 /2005 E6 " " 5 133957366a 133557283a 11/22/2005 H o fer (14 ) (04 EX P ) H o fe rxH arris o n (53) (04 E X P ) 12/19/2005 2 /17 /2005 E4 " " 6 N ew C lip # 1 133665491a 11/22/2005 H o fer 4-yr o ld H o fe rxH arris o n (46) (04 E X P ) 12/19/2005 2 /17 /2005 E3 " " 7 N ew C lip # 2 133779472a 11/22/2005 H o fer 4-yr o ld H o fe rxH arris o n (46) (04 E X P ) 12/19/2005 2 /17 /2005 E24 " " 8 N ew C lip # 3 133827615a 11/22/2005 H o fer 4-yr o ld H o fe rxH arris o n (46) (04 E X P ) . . - " " and R es earc h b ro o d 9 N ew C lip # 4 133966134a 11/22/2005 H o fer 4-yr o ld H o fe rxH arris o n (54) (04 E X P ) . . - " " and R es earc h b ro o d
10 N ew C lip # 5 133966134a 11/22/2005 H o fer 4-yr o ld H o fe rxH arris o n (54) (04 E X P ) . . - " " and R es earc h b ro o d 11 N ew C lip # 5 ad ipo s e c lip m ale 11/22/2005 H o fer 4-yr o ld H o fe r 4 -yr o ld 12/19/2005 2 /17 /2005 E20
12 134963680a 134931211a 11/30/2005 H o fer (13 ) (04 EX P ) H o fe rxH arris o n (48) (04 E X P ) . . - Utah 2 ,508 eggs 12-13-05 13 134929766a 133817266a 11/30/2005 H o ferxH arris o n (54) (04 EX P ) H o fe r (16) (04 E X P ) . . - and R es earc h B ro o d
14 134522140a C R R -24 12 /1/2005 C R R xH o fe r (35) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E2 G unnis o n R iv er p lant ? 15 133735465a C R R -22 12 /1/2005 C R R xH o fe r (25) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E10, P 1 (UC D A V IS ) 16 134512313a C R R -19 12 /1/2005 C R R xH o fe r (32) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E12, P 2 (UC D A VIS ) 17 134836296a C R R -21 12 /1/2005 C R R xH o fe r (36) (04 E XP ) G lenwo o d-C R R . . - 18 133661673a C R R -23 12 /1/2005 C R R xH o fe r (25) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E17, P 3 (UC D A VIS ) 19 134835230a C R R -20 12 /1/2005 C R R xH o fe r (43) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E18, P 4 (UC D A VIS ) 20 133662650a C R R -4 12 /1/2005 C R R xH o fe r (31) (04 E XP ) G lenwo o d-C R R 1/3/2005 3/7 /2006 E19, P 5 (UC D A VIS ) 21 134919273a C R R -8 12 /1/2005 C R R xH o fe r (36) (04 E XP ) G lenwo o d-C R R . . -
22 134963295a N c lip # 3 12/20/2005 H o ferxC R R (28) (04 E XP ) G lenwo o d-C R R . . - Y am pa R iv er 2 ,000 5-26-06 23 134616152a N c lip # 2 12/20/2005 H o ferxC R R (27) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 E22 S arv ic e C reek 24 133957222a N c lip # 4 12/20/2005 H o ferxC R R (11) (04 EX P ) G lenwo o d-C R R . . - and Yam pa R iv e r 5 ,500 5 -31-06 25 134961627a N c lip # 5 12/20/2005 H o ferxC R R (31) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 E25, W1 (UC D A VIS ) F ryingpan R iv er 4 ,430 5-17-06 26 133773235a N c lip # 6 12/20/2005 H o ferxC R R (2 ) (04 EX P ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W2 (UC D A V IS ) " " 27 133961091a N c lip # 7 12/20/2005 H o ferxC R R (31) (04 E XP ) G lenwo o d-C R R . . - " " 28 133877734a N c lip # 8 12/20/2005 H o ferxC R R (26) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W5 " " 29 133661243a N c lip # 9 12/20/2005 H o ferxC R R (32) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W6 " " 30 133672213a N c lip # 10 12/20/2005 H o ferxC R R (26) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W7, W 3 (UC D A VIS ) " " 31 133723393a N c lip # 11 12/20/2005 H o ferxC R R (1) (04 EX P ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W8, E 1 (UC D A V IS ) " " 32 134546717a N c lip # 12 12/20/2005 H o ferxC R R (31) (04 E XP ) G lenwo o d-C R R . . - " " 33 133764625a N c lip # 13 12/20/2005 H o ferxC R R (2 ) (04 EX P ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W9, W 4 and P 6 (UC D A VIS ) " " 34 134519513a N c lip # 14 12/20/2005 H o ferxC R R (1) (04 EX P ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W10 " " 35 134511753a N c lip # 15 12/20/2005 H o ferxC R R (30) (04 E XP ) G lenwo o d-C R R 1/18/2005 3 /21/2005 W11 " "
. . 36 C lip 36 N c lip # 16 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W12 R io G rande , 8 ,109 37 C lip 37 N c lip # 26 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W13 and B eav er C reek , 5 ,326 4 -20-06 38 C lip 38 N c lip # 18 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W14 " " 39 C lip 39 N c lip # 19 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W15 " " 40 C lip 40 N c lip # 20 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W16 " " 41 C lip 41 N c lip # 21 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W17 " " 42 C lip 42 N c lip # 22 12/21/2005 H o fer 4-yr o ld G lenwo o d-C R R 1/18/2005 3 /21/2005 W18 " "
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Table 2.2. Family groups created for M. cerebralis resistance laboratory testing (COOP wet lab brood) during spring 2006 spawning season.
Group Female Male Fertilized Descripton Female Origin Male Origin Location of eggs Hatched Dosed
. .
. . Elab Tank 12 133956316a Clip #6 12/1/2005 low x HoferxCRR (30) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 13 134877377a Clip #9 12/1/2005 med x HoferxCRR (37) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 17 133749392a Clip #12 12/1/2005 low HoferxCRR (24) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 18 133532116a Clip #18 12/1/2005 med HoferxCRR (33) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 19 133961134a Clip #17 12/1/2005 med HoferxCRR (20) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 21 133533450a Clip #11 12/1/2005 low x HoferxCRR (32) (04 EXP) Glenwood-CRR Quonset 12/27/2005 2/24/2005 Elab Tank 22 133962653a Clip #2 12/1/2005 low HoferxCRR (36) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 23 133547327a Clip # 13 12/1/2005 high x HoferxCRR (9) (04 EXP) Glenwood-CRR Quonset . . Elab Tank 25 133745327a Clip # 7 12/1/2005 low HoferxCRR (31) (04 EXP) Glenwood-CRR Quonset 12/27/2005 2/24/2005 Elab Tank 26 133533466a Clip #10 12/1/2005 med HoferxCRR (2) (04 EXP) Glenwood-CRR Quonset . .
CRR (G1) . . . Glenwood-CRR Glenwood-CRR Quonset 1/1/2006 3/7/2006 CRR (G2) . . . Glenwood-CRR Glenwood-CRR Quonset 1/1/2006 3/7/2006 CRR (G3) . . . Glenwood-CRR Glenwood-CRR Quonset 1/1/2006 3/7/2006
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Fish from each group were exposed to an average of 2,000 triactinomyxons per fish as two-month old fry. The fish were reared for five months post-exposure. Fish were fed a maintenance diet (Rangen trout feed, Rangen Inc., Buhl, Idaho) of roughly 2% body weight per day. Mortalities were removed and recorded daily. At the conclusion of the experiment, 10 fish were randomly selected from each family. The fish were measured and weighed, physical deformities were recorded, and heads were processed to enumerate myxospores per fish with the PTD (pepsin-trypsin digest) method.
Length, weight, and myxospore results were compared between strains using Proc
GLM in SAS system software. If significant differences were observed, Tukey's Studentized Range (HSD) test was used to determine which strains differed from each other. Alpha was set at 0.05 for all tests.
RESULTS
The GR rainbow trout developed the lowest spore counts of the groups tested, averaging 1,482 spores per fish (Figure 2.1). The CRR families developed the highest spore counts, averaging 232,973 spores per fish. The F1 families averaged 47,128 spores per fish. These results were similar to those found in the prior experiment. The B2 families developed higher spore counts, averaging 125,168 spores per fish. The statistical tests indicated that the CRR strain had significantly higher spore counts than the GR, F1 and B2 strains. The B2 strain had significantly higher spore counts than the GR strain, but not significantly higher than the F1 strain. The spore counts in the GR and F1 strains were not significantly different from each other. The GR, B2 and F1 strains averaged 15.3, 12.7, and 10.9 grams, respectively, at the end of the experiment. The pure CRR strain weighed significantly less than the GR strain at 7.7 grams. The pure CRR strain grew to an average of 87.3 mm, which was significantly shorter than the pure GR, B2, and F1 strains at 113.5, 108.4, and 105.5 mm, respectively.
DISCUSSION
In both the 2004 exposure experiment (Schisler et al. 2006) and this experiment, the F1 generation exhibited noticeable variation in spore counts and physical deformities between families. Within family variation in infection severity was relatively low. In this experiment, the B2 generation exhibited much more within family variation in infection severity (Figure 2.2). This is due to the re-assortment of genes and loss of resistance in some individual offspring of the B2 generation, but not of others. Only individuals inheriting resistance to whirling disease will be successful with regard to survival and reproductive potential in areas where the parasite has eliminated pure CRR populations. The rapid loss of resistance to M. cerebralis in subsequent generations of back-crosses in a hatchery setting could result in selection pressures that do not attain the goal of wild-strain fish with resistance to the parasite. Space constraints also limit the scope of this type of intensive selection in an artificial setting. An alternative to selecting families in a fish culture facility is to allow the selection among first generation crosses to occur in the wild. The selection pressure for individuals with both wild characteristics and resistance to M. cerebralis is immediate in locations where the parasite is endemic.
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Relatively good survival has been observed in first generation crosses in the wild (See Job 4, Whirling Disease Resistant Wild Strain Brood Stock Development and Evaluation). Therefore, it may be unnecessary to continue backcrossing F1 or B2 strains with pure CRR to ensure survival in the wild.
The level of resistance in the families and individuals created in this experiment
were compared with the previous generation of crosses. Genetic samples were collected from every individual used as parents in these lineages, and all of the offspring evaluated for resistance in these experiments. Comparison of the resistance in these fish with the genetic profiles of multiple families and individuals will help identify markers for resistance. Future research will investigate the continued heritability of whirling disease resistance, as well as physiological performance and survival of these crosses in the wild.
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Figure 2.1. Average spore counts for the three Hofer (GR), three Colorado River rainbow (CRR), ten F1 [GR-CRR (50:50)] and 16 B2 [GR-CRR (25:75)] strains. Each point represents average spore counts for each individual family.
0 100000 200000 300000 400000 500000 Myxospores
GR
CRR
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Figure 2.2. Example of inter-family variability in infection severity. Spore counts for two F1 [GR-CRR (50:50)] families and two B2 [GR-CRR (25:75)] families are shown. Ten fish per family were sampled. In this graph each point represents spore counts for each individual fish. Note that the B2 families show a large range of variation, from 0 to almost 400,000 spores, whereas the F1 families show a smaller range of variation, from 0 to only about 100,000 spores.
0 100000 200000 300000 400000 500000
Myxospores
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References
Hedrick, R. P., T. S. McDowell, G. D. Marty, G. T. Fosgate, K. Mukkatira, K. Myklebust, and M. El Matbouli. 2003. Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection. Diseases of Aquatic Organisms 55:37-44. Schisler, G. J. 2006. Salmonid Disease Studies. Federal Aid in Fish and Wildlife Restoration, Job Progress Report. Colorado Division of Wildlife, Fish Research Section. Fort Collins, Colorado. Schisler, G. J., K. A. Myklebust, and R. P. Hedrick. 2006. Inheritance of Myxobolus cerebralis resistance among F1-generation crosses of whirling disease resistant and susceptible rainbow trout strains. Journal of Aquatic Animal Health 18:109- 115.
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Experiment 2: Physiological characteristics and inheritance of Myxobolus cerebralis resistance among multiple generational crosses of the Hofer (GR) and Colorado River (CRR) rainbow trout strains.
INTRODUCTION
A laboratory experiment is currently being conducted at the Colorado Cooperative Fish and Wildlife Research Unit (COOP) wet lab in Fort Collins, Colorado to test the resistance of the German “Hofer” rainbow (GR) and Colorado River (CRR) rainbow trout strains, and crosses of these strains, to whirling disease. CRRs have historically been used for stocking in Colorado and they retain many of the desired wild rainbow trout characteristics needed to survive in Colorado’s rivers and streams. The CRR strain is, unfortunately, highly susceptible to whirling disease and their populations have experienced dramatic declines over the past decade. The GR strain has demonstrated very strong resistance to whirling disease in past exposure experiments. However, because the GR strain is a highly domesticated food fish, their survival and viability in the wild is uncertain. Also, the consequences of stocking this strain directly into the wild are unknown. In 2003, a breeding program was established to examine various crosses between the GR and CRR trout strains, with the ultimate goal of identifying those crosses that have the correct combination of resistant rainbow trout characteristics and wild rainbow trout characteristics to survive and reproduce in the wild in areas where heavy Myxobolus cerebralis infection exists. The resistance of two of these crosses, F1s and B2s, has been examined in previous exposure experiments (see previous section). F1s are the first filial generation cross between a pure GR individual and a pure CRR individual. B2s are the second generation backcross between an F1 individual and a pure CRR individual. These crosses were included in this exposure experiment to gain more knowledge about their inherited resistance to whirling disease. In addition, a third cross was included in this experiment to gain a better understanding of how resistant trout characteristics and wild trout characteristics are inherited in subsequent generations. This third cross is defined as true F2s, which are the second filial generation forward cross between two F1 individuals. The ultimate goal of this laboratory experiment is to further evaluate the resistance of the GR and CRR trout strains and their crosses to whirling disease, and to evaluate other characteristics that may play an important role in their survival in the wild including swimming performance and predator avoidance. Growth and feed efficiencies were also very closely monitored in this experiment.
METHODS Spawning of all families occurred at the Colorado Division of Wildlife Fish Research Hatchery (FRH) from mid-November 2006 through the end of January 2007. As in the previous experiment, both male and female pure GR and F1 fish are held on site
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and pure CRR individuals are obtained from the Colorado Division of Wildlife Glenwood Springs Hatchery (GWSH). F1 individuals, tagged with Passive Integrated Transponder (PIT) tags, were identified by their 10 digit alpha numeric code (Table 2.1). An anal fin clip was taken from each spawned individual for later genetic analysis. Eggs were placed in incubators at the FRH or COOP wet lab and held until they were eyed (Table 2.1). Once eyed, picked eggs were placed in 20 gallon (76 liter) tanks containing short (7 cm) standpipes for a greater amount of water turnover at the COOP wet lab, where they were hatched. Upon swim-up, tall (30 cm) standpipes were placed in the tanks and the fish were started on size 0 trout diet. After approximately 335 degree days (ºC), fish were fed size 1 trout diet (Table 2.3). At this time, families were reduced to 50 fish per family. This was considered the beginning of the growth experiment. Each family was batch weighed and fed 4% of the total fish weight. Families were maintained at 50 fish until the day before infection in order to account for any mortality that may have occurred as a result of the switch to a larger feed size. An additional 50 fish from four of the families from each strain were removed and placed in uninfected control tanks (Table 2.2). At the time of infection, families were reduced to 25 fish. Again, fish were batch weighed and fed 4% of the total fish weight (Table 2.3). Control families were also reduced to 25 fish at this time. Fish were reweighed every two weeks and feed amount was changed accordingly. Fish were switched to size 2 trout diet at a batch weight of 75 grams, size 3 trout diet at a batch weight of 162.5 grams, and size 4 trout diet at a batch weight of 500 grams, according to hatchery trout feed guidelines (Tables 2.4 and 2.5). Fish were infected at an average of 678 degree days (ºC) post-hatch (Table 2.1). Triactinomyxons (TAMs) for exposures beginning on February 15, 2007 and continuing through April 20, 2007 originated at Ron Hedrick’s lab at U.C. Davis. TAMs for exposures after April 20, 2007 came from Barry Nehring with the Colorado Division of Wildlife in Montrose, Colorado. Cultures of TAMs in both cases were produced from Mt. Whitney Tubifex tubifex worms. TAMs were counted by mixing 1,000 μl of filtrate containing the TAMs and 60 μl of crystal violet used to dye the TAMs to make them easier to see; 84.6 μl of this mixture was then placed on a slide and TAMs per slide were counted. Ten counts were conducted on the filtrates prior to exposures to increase confidence in estimates of TAM concentrations in the filtrate. An average of the counts was taken, and this number was used to calculate the amount of TAMs per ml. Fish were infected with 2,000 TAMs per individual, a total of 50,000 TAMs per tank. Before exposure, the water flow to each aquarium was halted and each aquarium received copious aeration with an air stone to ensure full mixing of the TAMs and equal exposure of all fish. The approximate amount of filtrate to deliver 2,000 TAMs per fish was measured out, placed in a 1,000 ml beaker, and evenly distributed throughout each aquarium. This was done in two passes to ensure equal distribution of TAMs in the tank and to account for a possible unequal distribution of TAMs within the filtrate. Water flow was halted for one hour to ensure complete infection of all fish. Swimming experiments began on April 9, 2007. Five fish were randomly chosen from four randomly chosen families of each strain to be swum at two weeks, one month,
15
and two and a half months post-exposure in two Loligo® swimming flumes. Fish were marked before being swam with Northwest Marine Technologies fluorescent visual implant elastomer (VIE) tags so that the same fish could be swam at all three dates. After a fish was placed in the flume, it was run at the lowest speed of 2 cm/s for one hour. This allowed the fish to acclimate to the flume and recover from handling. After the one hour acclimation period, the flume speed was increased to 5 cm/s for ten minutes. Flume speed continued to be increased by 5 cm/s every ten minutes until the fish reached its critical swimming velocity (CSV). The fish was considered to have reached its CSV when it was no longer swimming against the current and was pushed up against the screen at the back of the flume. The fish was then removed from the flume, measured and weighed, and allowed to recover in an aerated bucket held at the same temperature as the flume. A total of 535 fish will be swum over the course of this experiment. The goal of the swimming experiment is determine differences in CSVs between the strains, and to determine whether there is a difference in swimming ability between infected and non- infected fish within a strain. Swimming experiments will continue through mid-August 2007. The exposure experiment will conclude once the fish have reached approximately 2,000 degree days (ºC) post-exposure, which is approximately five months post-exposure. Ten fish will be randomly removed from each tank, measured and weighed. The head of each individual will then be removed and cut in half from the nose to the back of the head. Half of the head will be used for histological analysis, and the other half will be used to determine spore load using pepsin tripsin digest (PTD) testing. In addition, the body of each individual will be kept and used to determine an average total lipid count for each strain. Following the conclusion of the exposure experiment, behavioral experiments will also be conducted using the remainder of the fish in each family. The goal of the behavior experiment is to determine the ability of a strain, as well as infected and non-infected fish within a strain, to detect and avoid predators.
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Table 2.3. Family groups created for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007. Group Strain Male Female Male Origin Female Origin Spawn Date Location of Eggs Status Hatched Dosed Number Degree Days Tank
RH15 B2 (CRR x F1) M15 F3 - 133653755A GWSH Group 35 12/12/2006 FRH Original 1/9/2007 3/21/2007 25 629.30 40
RH19 B2 (CRR x F1) M20 F4 - 134836296A GWSH Group 36 12/12/2006 FRH Original Dead as eggs
RH48 B2 (CRR x F1) M48 F36 - 134616152A GWSH Group 27 1/11/2007 FRH Replaced RH19 2/8/2007 4/13/2007 25 668.3 68
QT71 B2 (F1 x CRR) M31 - 133752472A F53 Group 43 GWSH 1/17/2007 COOP Original 3/6/2007 5/4/2007 25 616.20 71
RH101 B2 (F1 x CRR) M41 - 134936464 F63 Group 30 GWSH 1/30/2007 FRH Extras Not Needed
QT61 CRR M54 F44 GWSH GWSH 1/17/2007 COOP Original 3/14/2007 5/29/2007 25 762.70 28
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Table 2.3. (continued). Family groups created for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007. Group Strain Male Female Male Origin Female Origin Spawn Date Location of Eggs Status Hatched Dosed Number Degree Days Tank
RH85 CRR M64 F56 GWSH GWSH 1/30/2007 FRH Extras Not Needed
RH21 F1 (CRR x GR) M22 F13 GWSH BFRH - RW9 12/12/2006 FRH Original 1/9/2007 3/21/2007 25 629.30 13
RH22 F1 (CRR x GR) M23 F14 GWSH BFRH - RW9 12/12/2006 FRH Original Dead as eggs
RH56 F1 (CRR x GR) M49 F39 GWSH BFRH - RW9 1/11/2007 FRH Replaced RH22 2/12/2007 4/27/2007 25 691.20 11
RH57 F1 (CRR x GR) M50 F40 GWSH BFRH - RW9 1/11/2007 FRH Extras Not Needed
RH59 F1 (CRR x GR) M52 F42 GWSH BFRH - RW9 1/11/2007 FRH Extras Dead as eggs
RH100 F1 (GR x CRR) M79 F70 BFRH - RW6 GWSH 1/30/2007 FRH Replaced RH93 2/27/2007 5/1/2007 25 679.30 52
RH88 F1 (GR x CRR) M67 F58 BFRH - RW6 GWSH 1/30/2007 FRH Original 2/26/2007 5/11/2007 25 734.20 9
RH89 F1 (GR x CRR) M68 F59 BFRH - RW6 GWSH 1/30/2007 FRH Original Dead as eggs
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RH2 F1 x B2 M2 - 146147570A F2 - 134521446A Group 14 Group 11 11/28/2006 FRH Extras Not Needed
RH1 F2 M1 - 133874590A F1 - 133735465A Group 30 Group 25 11/21/2006 FRH Original 12/19/2006 2/15/2007 25 640.40 49
RH47 F2 M47 - 133736183A F35 - 134963295A Group 36 Group 28 12/28/2006 FRH Original Dead as eggs
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RH81 F2 M46 - 133611735A F54 - 134961627A Group 1 Group 31 1/17/2007 FRH Replaced RH47 2/17/2007 4/20/2007 25 639.80 51
RH82 F2 M44 - 134757611A F54 - 134961627A Group 36 Group 31 1/17/2007 FRH Extras Not Needed
RH10 GR M10 F7 BFRH - RW2 BFRH - RW6 11/29/2006 FRH Original 12/27/2006 3/6/2007 25 640.40 22
RH33 GR M33 F25 BFRH - RW6 BFRH - RW9 12/13/2006 FRH Original Dead as eggs
RH34 GR M34 F26 BFRH -RW6 BFRH - RW9 12/13/2006 FRH Original 1/10/2007 3/13/2007 25 668.20 50
RH36 GR M36 F28 BFRH - RW6 BFRH - RW9 12/13/2006 FRH Replaced RH33 1/11/2007 4/11/2007 25 797.8 45
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Table 2.4. Control groups separated from family groups created for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007. Group Strain Male Female Male Origin Female Origin Spawn Date Location of Eggs Status Hatched Dosed Number Degree Days Tank
RH8 GR M8 F5 BFRH - RW2 BFRH - RW6 11/29/2006 FRH Original 12/27/2006 N/A 25 N/A C1
RH11 GR M11 F8 BFRH - RW2 BFRH - RW6 11/29/2006 FRH Original 12/27/2006 N/A 25 N/A C14
RH34 GR M34 F26 BFRH -RW6 BFRH - RW9 12/13/2006 FRH Original 1/10/2007 N/A 25 N/A C4
RH36 GR M36 F28 BFRH - RW6 BFRH - RW9 12/13/2006 FRH Replaced RH33 1/11/2007 N/A 25 N/A C11
QT68 CRR M61 F51 GWSH GWSH 1/17/2007 COOP Original 3/5/2007 N/A 25 N/A C15
RH27 F1 (CRR x GR) M30 F19 GWSH BFRH - RW9 12/12/2006 FRH Original 1/9/2007 N/A 25 N/A C19
RH30 F1 (CRR x GR) M34 F22 GWSH BFRH - RW9 12/12/2006 FRH Original 1/9/2007 N/A 25 N/A C20
RH92 F1 (GR x CRR) M71 F62 BFRH - RW6 GWSH 1/30/2007 FRH Original 2/27/2007 N/A 25 N/A C17
RH97 F1 (GR x CRR) M76 F67 BFRH - RW6 GWSH 1/30/2007 FRH Original 2/27/2007 N/A 25 N/A C2
RH1 F2 M1 - 133874590A F1 - 133735465A Group 30 Group 25 11/21/2006 FRH Original 12/19/2006 N/A 25 N/A C9
QT78 B2 (F1 x CRR) M5 - 133669695A F45 Group 11 GWSH 1/17/2007 COOP Original 3/5/2007 N/A 25 N/A C5
QT72 B2 (F1 x CRR) M45 - 135126471A F54 Group 1 GWSH 1/17/2007 COOP Original 3/13/2007 N/A 25 N/A C8
RH16 B2 (CRR x F1) M17 F10 - 133957222A GWSH Group 11 12/12/2006 FRH Original 1/9/2007 N/A 25 N/A C12
RH17 B2 (CRR x F1) M18 F11 - 133661673A GWSH Group 25 12/12/2006 FRH Original 1/9/2007 N/A 25 N/A C18
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Table 2.5. Batch weights and feed amounts for families on size 1 Rangen trout diet for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007. Tank # Group Strain Date on Size 1 DD @ Size 0 # Fish Batch Weight Feed 4% (grams) Date Reduced # Fish Batch Weight Feed 4% (grams)
40 RH15 B2 CRR(m) x F1(f) 3/3/2007 327.7 50 12 0.48 3/20/2007 25 10 0.4
63 RH16 B2 CRR(m) x F1(f) 2/25/2007 347.4 50 22 0.88 3/12/2007 25 18 0.72
C12 RH16 B2 CRR(m) x F1(f) 2/25/2007 347.4 50 22 0.88 3/12/2007 25 20 0.8
3 RH17 B2 CRR(m) x F1(f) 3/3/2007 327.7 50 21 0.84 3/20/2007 25 15 0.6
C18 RH17 B2 CRR(m) x F1(f) 3/3/2007 327.7 50 21 0.84 3/20/2007 25 17 0.68
16 RH18 B2 CRR(m) x F1(f) 3/3/2007 327.7 50 18 0.72 3/20/2007 25 15 0.6
55 RH20 B2 CRR(m) x F1(f) 2/25/2007 347.4 50 13 0.52 3/12/2007 25 12 0.48
66 RH37 B2 CRR(m) x F1(f) 3/11/2007 349.2 50 15 0.6 3/29/2007 25 15 0.6
30 RH39 B2 CRR(m) x F1(f) 3/15/2007 331.5 50 21 0.84 4/5/2007 25 20 0.8
32 RH41 B2 CRR(m) x F1(f) 3/15/2007 331.5 50 15 0.6 4/5/2007 25 15 0.6
41 RH43 B2 CRR(m) x F1(f) 3/15/2007 331.5 48 23 0.92 4/5/2007 25 21 0.84
68 RH48 B2 CRR(m) x F1(f) 4/3/2007 383.2 50 18 0.72 4/10/2007 25 12 0.48
71 QT71 B2 F1(m) x CRR(f) 4/30/2007 352.4 50 17 0.68 5/3/2007 25 9 0.36
8 QT72 B2 F1(m) x CRR(f) 5/13/2007 358 50 19 0.76 5/24/2007 25 15 0.6
C8 QT72 B2 F1(m) x CRR(f) 5/13/2007 358 50 18 0.72 5/24/2007 25 14 0.56
76 QT73 B2 F1(m) x CRR(f) 4/30/2007 342.2 50 17 0.68 5/3/2007 25 10 0.4
35 QT74 B2 F1(m) x CRR(f) 5/13/2007 358 50 23 0.92 5/29/2007 25 20 0.8
74 QT75 B2 F1(m) x CRR(f) 5/1/2007 333.2 50 15 0.6 5/3/2007 25 8 0.32
36 QT76 B2 F1(m) x CRR(f) 5/13/2007 358 50 21 0.84 5/24/2007 25 16 0.64
59 QT77 B2 F1(m) x CRR(f) 4/30/2007 342.2 50 18 0.72 5/7/2007 25 11 0.44
57 QT78 B2 F1(m) x CRR(f) 4/30/2007 352.4 50 21 0.84 5/3/2007 25 12 0.48
C5 QT78 B2 F1(m) x CRR(f) 4/30/2007 352.4 50 21 0.84 5/3/2007 25 12 0.48
69 QT79 B2 F1(m) x CRR(f) 5/1/2007 333.2 50 17 0.68 5/3/2007 25 11 0.44
31 QT80 B2 F1(m) x CRR(f) 5/17/2007 363.1 50 16 0.64 5/29/2007 25 14 0.56
28 QT61 CRR 5/13/2007 358 50 17 0.68 5/29/2007 25 15 0.6
78 QT62 CRR 4/30/2007 352.4 50 17 0.68 5/7/2007 25 10 0.4
C16 QT62 CRR 4/30/2007 352.4 50 18 0.72 5/7/2007 25 10 0.4
27 QT63 CRR 5/13/2007 358 50 18 0.72 5/29/2007 25 16 0.64
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Table 2.5. (continued). Batch weights and feed amounts for families on size 1 Rangen trout diet for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007. Tank # Group Strain Date on Size 1 DD @ Size 0 # Fish Batch Weight Feed 4% (grams) Date Reduced # Fish Batch Weight Feed 4% (grams)
C7 QT63 CRR 5/13/2007 358 50 18 0.72 5/29/2007 25 16 0.64
47 QT64 CRR 5/17/2007 363.1 50 16 0.64 5/29/2007 25 11 0.44
15 QT65 CRR 5/13/2007 358 50 17 0.68 5/24/2007 25 13 0.52
39 QT66 CRR 5/13/2007 358 50 18 0.72 5/24/2007 25 13 0.52
79 QT67 CRR 4/30/2007 352.4 50 16 0.64 5/3/2007 25 9 0.36
80 QT68 CRR 4/30/2007 352.4 50 17 0.68 5/3/2007 25 11 0.44
C15 QT68 CRR 4/30/2007 352.4 50 18 0.72 5/3/2007 25 10 0.4
56 QT69 CRR 5/1/2007 333.2 50 17 0.68 5/3/2007 25 9 0.36
5 QT70 CRR 5/8/2007 341.2 50 18 0.72 5/24/2007 25 16 0.64
C10 QT70 CRR 5/8/2007 341.2 50 18 0.72 5/24/2007 25 14 0.56
13 RH21 F1 CRR(m) x HOF(f) 3/3/2007 327.7 50 21 0.84 3/20/2007 25 17 0.68
33 RH24 F1 CRR(m) x HOF(f) 3/3/2007 327.7 37 19 0.76 3/20/2007 25 20 0.8
61 RH25 F1 CRR(m) x HOF(f) 2/25/2007 347.4 50 21 0.84 3/12/2007 25 20 0.8
54 RH26 F1 CRR(m) x HOF(f) 2/25/2007 347.4 50 22 0.88 3/12/2007 25 18 0.72
2 RH27 F1 CRR(m) x HOF(f) 3/3/2007 327.7 50 21 0.84 3/20/2007 25 18 0.72
C19 RH27 F1 CRR(m) x HOF(f) 3/3/2007 327.7 50 22 0.88 3/20/2007 25 20 0.8
48 RH28 F1 CRR(m) x HOF(f) 3/3/2007 327.7 27 15 0.6 3/20/2007 25 20 0.8
37 RH30 F1 CRR(m) x HOF(f) 3/3/2007 327.7 50 22 0.88 3/20/2007 25 17 0.68
C20 RH30 F1 CRR(m) x HOF(f) 3/3/2007 327.7 50 22 0.88 3/20/2007 25 18 0.72
11 RH56 F1 CRR(m) x HOF(f) 4/4/2007 338.6 50 22 0.88 4/26/2007 25 24 0.96
14 RH58 F1 CRR(m) x HOF(f) 4/4/2007 338.6 50 23 0.92 4/23/2007 25 24 0.96
25 RH60 F1 CRR(m) x HOF(f) 4/4/2007 338.6 50 20 0.8 4/23/2007 25 17 0.68
52 RH100 F1 HOF(m) x CRR(f) 4/19/2007 346.7 50 19 0.76 4/30/2007 25 14 0.56
9 RH88 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 23 0.92 5/10/2007 25 22 0.88
20 RH91 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 20 0.8 5/10/2007 25 19 0.76
62 RH92 F1 HOF(m) x CRR(f) 4/22/2007 358.1 50 30 1.2 4/30/2007 25 21 0.84
C17 RH92 F1 HOF(m) x CRR(f) 4/22/2007 358.1 50 28 1.12 4/30/2007 25 20 0.8
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38 RH94 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 18 0.72 5/10/2007 25 18 0.72
34 RH95 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 15 0.6 5/10/2007 25 12 0.48
73 RH96 F1 HOF(m) x CRR(f) 4/19/2007 346.7 50 19 0.76 4/30/2007 25 13 0.52
44 RH97 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 24 0.96 5/10/2007 25 21 0.84
C2 RH97 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 24 0.96 5/10/2007 25 20 0.8
64 RH98 F1 HOF(m) x CRR(f) 4/22/2007 358.1 50 16 0.64 4/30/2007 25 11 0.44
17 RH99 F1 HOF(m) x CRR(f) 4/23/2007 333.6 50 19 0.76 5/10/2007 25 16 0.64
44 RH2 F1xB2 2/14/2007 338.5 50 23 0.92 N/A - Killed N/A N/A N/A
49 RH1 F2 F1(m) x F1(f) 1/30/2007 337.2 50 19 0.76 2/14/2007 25 24 0.96
C9 RH1 F2 F1(m) x F1(f) 1/30/2007 337.2 50 20 0.8 2/14/2007 25 19 0.76
65 RH13 F2 F1(m) x F1(f) 2/18/2007 340.5 50 18 0.72 3/5/2007 25 15 0.6
67 RH14 F2 F1(m) x F1(f) 2/18/2007 340.5 50 19 0.76 3/5/2007 25 17 0.68
C6 RH14 F2 F1(m) x F1(f) 2/18/2007 340.5 50 20 0.8 3/5/2007 25 19 0.76
7 RH3 F2 F1(m) x F1(f) 2/14/2007 338.5 50 22 0.88 3/5/2007 26 17 0.68
C3 RH3 F2 F1(m) x F1(f) 2/14/2007 338.5 50 23 0.92 3/5/2007 25 20 0.8
46 RH38 F2 F1(m) x F1(f) 4/15/2007 344.8 50 19 0.76 4/23/2007 25 10 0.4
12 RH40 F2 F1(m) x F1(f) 4/15/2007 344.8 50 22 0.88 4/23/2007 25 14 0.56
6 RH42 F2 F1(m) x F1(f) 4/4/2007 338.6 50 14 0.56 4/19/2007 25 12 0.48
21 RH45 F2 F1(m) x F1(f) 4/12/2007 341.5 50 14 0.56 4/23/2007 25 9 0.36
77 RH46 F2 F1(m) x F1(f) 3/23/2007 334.1 50 19 0.76 4/2/2007 25 14 0.56
72 RH49 F2 F1(m) x F1(f) 4/14/2007 330.7 50 12 0.48 4/16/2007 25 7 0.28
4 RH5 F2 F1(m) x F1(f) 2/14/2007 338.5 50 20 0.8 3/5/2007 25 15 0.6
C13 RH5 F2 F1(m) x F1(f) 2/14/2007 338.5 50 19 0.76 3/5/2007 25 19 0.76
18 RH50 F2 F1(m) x F1(f) 4/23/2007 333.6 50 18 0.72 5/7/2007 25 15 0.6
26 RH51 F2 F1(m) x F1(f) 4/30/2007 343.2 50 14 0.56 5/7/2007 25 9 0.36
53 RH52 F2 F1(m) x F1(f) 4/10/2007 331.4 50 17 0.68 4/16/2007 25 11 0.44
24
1 RH53 F2 F1(m) x F1(f) 4/30/2007 343.2 50 20 0.8 5/3/2007 25 12 0.48
43 RH54 F2 F1(m) x F1(f) 5/3/2007 346.3 50 17 0.68 5/11/2007 25 12 0.48
29 RH55 F2 F1(m) x F1(f) 4/30/2007 343.2 50 16 0.64 5/10/2007 25 12 0.48
42 RH6 F2 F1(m) x F1(f) 2/14/2007 338.5 50 21 0.84 3/5/2007 25 19 0.76
51 RH81 F2 F1(m) x F1(f) 4/14/2007 330.7 50 10 0.4 4/19/2007 25 5 0.2
24 RH83 F2 F1(m) x F1(f) 4/30/2007 343.2 50 12 0.48 5/11/2007 24 9 0.36
22 RH10 GR 2/14/2007 338.5 50 19 0.76 3/5/2007 25 15 0.6
60 RH11 GR 2/8/2007 335.9 50 18 0.72 2/22/2007 25 18 0.72
C14 RH11 GR 2/8/2007 335.9 50 20 0.8 2/22/2007 25 20 0.8
23 RH12 GR 2/14/2007 338.5 50 22 0.88 3/5/2007 25 19 0.76
70 RH31 GR 3/1/2007 329.6 50 14 0.56 3/12/2007 25 11 0.44
75 RH32 GR 3/3/2007 329.3 50 30 1.2 3/12/2007 25 21 0.84
50 RH34 GR 3/1/2007 329.6 50 14 0.56 3/12/2007 25 11 0.44
C4 RH34 GR 3/1/2007 329.6 50 13 0.52 3/12/2007 25 9 0.36
19 RH35 GR 3/11/2007 331 50 14 0.56 4/10/2007 25 19 0.76
45 RH36 GR 3/15/2007 334.1 50 13 0.52 4/10/2007 25 16 0.64
C11 RH36 GR 3/15/2007 334.1 50 13 0.52 4/10/2007 25 16 0.64
58 RH8 GR 2/8/2007 335.9 50 21 0.84 2/22/2007 25 19 0.76
C1 RH8 GR 2/8/2007 335.9 50 20 0.8 2/22/2007 25 21 0.84
10 RH9 GR 2/14/2007 338.5 50 22 0.92 3/5/2007 25 19 0.76
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Table 2.6. Batch weights and feed amounts for families on size 2 Rangen trout diet for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007.
Tank # Group Strain Date on Size 2 DD @ Size 1 # Fish Grams Feed 4% (grams) C12 RH16 B2 CRR(m) x F1(f) 5/7/2007 727.6 25 89 3.56 63 RH16 B2 CRR(m) x F1(f) 5/7/2007 727.6 25 81 3.24
C18 RH17 B2 CRR(m) x F1(f) 5/15/2007 751.6 25 92 3.68 3 RH17 B2 CRR(m) x F1(f) 5/15/2007 682.5 25 83 3.32
30 RH39 B2 CRR(m) x F1(f) 5/17/2007 605 25 80 3.2 13 RH21 F1 CRR(m) x HOF(f) 5/15/2007 682.5 25 99 3.96 33 RH24 F1 CRR(m) x HOF(f) 5/15/2007 682.5 25 98 3.92 61 RH25 F1 CRR(m) x HOF(f) 4/23/2007 590.9 25 88 3.52 54 RH26 F1 CRR(m) x HOF(f) 5/7/2007 727.6 25 97 3.88
C19 RH27 F1 CRR(m) x HOF(f) 5/1/2007 605.8 24 82 3.28 2 RH27 F1 CRR(m) x HOF(f) 5/15/2007 682.5 25 103 4.12
48 RH28 F1 CRR(m) x HOF(f) 5/15/2007 682.5 25 110 4.4 C20 RH30 F1 CRR(m) x HOF(f) 5/1/2007 605.8 24 76 3.04 37 RH30 F1 CRR(m) x HOF(f) 5/15/2007 682.5 25 89 3.56 49 RH1 F2 F1(m) x F1(f) 4/12/2007 740.5 25 97 3.88 C9 RH1 F2 F1(m) x F1(f) 4/12/2007 740.5 25 106 4.24 65 RH13 F2 F1(m) x F1(f) 4/30/2007 727 25 106 4.24 67 RH14 F2 F1(m) x F1(f) 4/16/2007 574.7 24 79 3.16 C6 RH14 F2 F1(m) x F1(f) 4/16/2007 574.7 25 89 3.56 C3 RH3 F2 F1(m) x F1(f) 4/30/2007 767.4 25 96 3.84 7 RH3 F2 F1(m) x F1(f) 5/14/2007 804.6 26 85 3.4
C13 RH5 F2 F1(m) x F1(f) 4/30/2007 767.4 25 100 4 4 RH5 F2 F1(m) x F1(f) 5/14/2007 804.6 25 84 3.36
42 RH6 F2 F1(m) x F1(f) 4/30/2007 661.4 25 95 3.8 22 RH10 GR 4/30/2007 661.4 25 104 4.16 60 RH11 GR 4/6/2007 586.5 25 92 3.68
C14 RH11 GR 4/6/2007 586.5 25 115 4.6 23 RH12 GR 4/16/2007 525.5 25 90 3.6
26
Table 2.6. (continued). Batch weights and feed amounts for families on size 2 Rangen trout diet for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007.
Tank # Group Strain Date on Size 2 DD @ Size 1 # Fish Grams Feed 4% (grams) 70 RH31 GR 5/7/2007 687.1 24 99 3.96 75 RH32 GR 4/23/2007 530.5 25 108 4.32 50 RH34 GR 5/7/2007 687.1 24 99 3.96 C4 RH34 GR 5/7/2007 687.1 19 81 3.24 58 RH8 GR 4/6/2007 586.5 25 103 4.12 C1 RH8 GR 4/6/2007 586.5 25 122 4.88 10 RH9 GR 4/16/2007 525.5 25 78 3.12
Table 2.7. Batch weights and feed amounts for families on size 3 Rangen trout diet for M. cerebralis resistance experiment conducted at the Colorado Cooperative Fish and Wildlife Unit (COOP) wet lab in 2006-2007.
Tank # Group Strain Date on Size 3 DD @ Size 2 # Fish Grams Feed 4% (grams) 49 RH1 F2 F1(m) x F1(f) 5/10/2007 296.9 25 169 6.76 C9 RH1 F2 F1(m) x F1(f) 5/10/2007 296.9 25 181 7.24 C6 RH14 F2 F1(m) x F1(f) 5/14/2007 298 25 178 7.12 22 RH10 GR 5/14/2007 143.2 25 170 6.8
C14 RH11 GR 4/20/2007 145.5 25 190 7.6 60 RH11 GR 5/4/2007 294.4 25 234 9.36 23 RH12 GR 5/14/2007 279.1 25 208 8.32 75 RH32 GR 5/7/2007 146.7 25 178 7.12 C1 RH8 GR 4/20/2007 145.5 25 193 7.72 58 RH8 GR 5/4/2007 294.4 25 243 9.72 10 RH9 GR 5/14/2007 279.1 25 207 8.28
27
Job No. 3: Whirling Disease Resistant Domestic Brood Stock Development and Evaluation
Job Objective: These experiments are focused on the performance of the Hofer (GR)
strain and GR-Harrison strain as domestic production fish compared with other commonly used production fish.
Hatchery Performance Evaluations: Performance of a whirling disease resistant rainbow trout strain at two Myxobolus cerebralis-positive trout rearing facilities.
ABSTRACT
A recently identified strain of rainbow trout with resistance to whirling disease (GR) was compared with Tasmanian and Bellaire rainbow trout strains in two separate trout rearing facilities to evaluate its performance and susceptibility to M. cerebralis infection under standard rearing conditions. Fish were brought to the facilities as either advanced fingerlings or as eyed eggs. Growth in the GR strain was significantly faster than in these other two domestic strains. Infection severity and prevalence in the GR strain was significantly lower than in the other two strains. These results demonstrate that the GR strain may be a useful replacement for more susceptible strains in facilities with a history of M. cerebralis infection.
INTRODUCTION Whirling disease, caused by Myxobolus cerebralis is known to cause severe declines in wild rainbow trout populations, particularly in the Intermountain West (Nehring and Walker 1996, Vincent 1996). The parasite has become established in many fish culture facilities as well. For example, fish in 10 of Colorado’s 14 state-operated trout rearing facilities were identified as infected with the parasite as recently as 1997 (Rich Kolecki, Colorado Division of Wildlife Chief of Hatcheries, personal communication). While infections from the parasite in hatchery situations do not typically result in heavy mortality, other detrimental effects such as compromised growth, performance, and conformation of the infected fish can occur. This can result in reduced marketability of the fish in commercial operations. Possible spread of the parasite from infected facilities can also be a damaging consequence. Spread of M. cerebralis through human transfer of infected fish is well documented, and considered to be one of the primary routes of dispersal (Hoffmann 1990, Modin 1998, Bartholomew and Reno 2002). Stocking of infected fish has been shown to increase the likelihood of M. cerebralis establishment (Schisler 2002), and greatly increase the ambient parasite load and infection severity in fish in the near vicinity and downstream of the stocked locations (Nehring 2006). In some states, regulations require that facilities harboring the parasite be depopulated and the parasite eliminated from the water supply or the facilities be closed.
28
Fish culture problems related to whirling disease infection can be alleviated in many cases through improved management practices to reduce or eliminate the parasite (Hoffman 1990). Solutions include using well water or water treatment to ensure parasite-free water supplies. Hatchery renovation, such as installation of concrete raceways or lining earthen ponds can also help eliminate habitat for the intermediate host, Tubifex tubifex. These practices can greatly reduce the incidence and prevalence of infection in some locations. In Colorado, hatchery improvements have eliminated the parasite from seven of the facilities previously identified as positive for the parasite. The parasite cannot be eliminated in some facilities because of reliance on infected water sources. In these situations, hatchery managers may be somewhat limited in their ability to reduce infection prevalence and severity.
Many previous studies have demonstrated that rainbow trout are quite vulnerable
to whirling disease (Thompson et. al 1999, Hedrick et al. 1999, Vincent 2002), and until recently, all rainbow trout strains were considered to be very susceptible to the parasite. The discovery of whirling disease-resistant rainbow trout strains (Hedrick et al. 2003, Schisler et al. 2006, Wagner et al. 2006) has provided hope that effects of the parasite could be further alleviated through the use of these resistant strains in trout rearing facilities where M. cerebralis cannot be completely eradicated. The potential use of these strains as a method to reduce impacts due to M. cerebralis has generated considerable interest. Performance of the GR strain in typical fish culture situations in the United States has not yet been evaluated, and verification of the resistance of these strains to whirling disease under normal culture conditions is needed to determine if their use is a viable option. This study was designed to evaluate the growth and survival of the GR strain when compared with other standard domestic strains in representative hatchery situations.
METHODS
GR strain rainbow trout were evaluated at two separate state-operated M.
cerebralis-positive trout rearing facilities. Both of these facilities rely on surface water, and have a history of infection in fish reared at these locations. Bellaire strain and Tasmanian strain rainbow trout are commonly used in Colorado as a catchable rainbow trout product for put-and-take and put-grow-and-take fisheries. In both of the trials described herein, the Bellaire and Tasmanian strain lots were reared through their normal production cycle, and matched with equal numbers of the GR strain to compare the growth and infectivity between the strains. Chalk Cliffs Rearing Unit. – The Chalk Cliffs Rearing Unit is located in the upper Arkansas River drainage near Nathrop, Colorado, at an elevation of 2,438 meters. The facility was first identified as positive for M. cerebralis in March, 1988. The facility relies on surface water from Chalk Creek, and fish are reared in a series of raceways and earthen ponds. Warm springs in Chalk Creek result in an increased ambient temperature through the winter months (Figure 3.1). Myxospore counts in fish collected from the ponds on the facility during annual disease inspections have at times averaged over one million per fish. Improved management practices such as regular removal of moralities
29
and rotation of active ponds, with periodic drying and excavating, have helped reduce myxospore counts in recent years. However, because of its reliance on surface water, the Chalk Cliffs facility cannot be completely rid of the parasite.
Eyed eggs of the GR and Tasmanian strain rainbow trout were transported to the facility in December, 2005. The eggs hatched within a day of each other, and fry were reared together in 0.2 x 3.5 m troughs in a hatchery building, fed with 38-53 liters per minute surface water. At six months post-hatch, the fish were moved to 1 x 50 m raceways with a flow of 4,920 liters per minute for further growth, then to a 0.47 hectare pond for final grow-out at 11 months post-hatch. Growth was measured periodically throughout the rearing period, starting at four months post-hatch, by using wet weights. Direct length measurements for statistical comparisons were made at nine and a half months and one year post-hatch.
Samples were collected to test for M. cerebralis infection and prevalence at three
months post-hatch (1,002 degree-days ºC). Ten fish from each lot were collected and euthanized with tricaine methanesulfonate for histological analysis. Because of the size of the fish, they were fixed whole in Davidson’s solution for 48 hours and then transferred to 70% ethanol. The bodies were embedded in paraffin, sectioned and stained with hematoxylin and eosin by standard procedures (Humason 1979). Two sections, one 30 microns deeper than the other, for each fish were evaluated for the presence of microscopic lesions due to M. cerebralis. The severity of microscopic lesions present in stained tissue sections were evaluated by the MacConnell-Baldwin scale using a scale from 0 – 5 with 5 representing the most severe lesions and 0 indicating no abnormalities seen (Hedrick et al. 1999b, Baldwin et al. 2000).
At five months post-hatch (1,934 degree-days ºC), ten fish of each strain were
again collected for histological analysis and 10 fish of each strain were collected for PTD analysis. Heads were removed from the sample fish. Whole heads designated for histological sectioning were preserved in Davidson’s solution. Histological procedures were conducted as described above. If used for PTD analysis, heads were placed in individually labeled plastic bags, and then held at – 20oC until processing. The samples were then soaked in water at 45°C to soften the tissues, and then skeletal elements were separated from soft tissue by agitation in a wrist-action electric shaker using glass marbles as hammers. The samples were then decanted through disposable 190 µm calculi filters and rinse water was added back to the skeletal elements for purification and concentration by PTD (Markiw and Wolf 1974) and myxospore quantification (O’Grodnick 1975).
A third sample was collected during the facilities annual disease inspection, at
nine months post-hatch (3,468 degree-days ºC). This collection occurred after the fish had been in the raceways for three and a half months (1,402 degree-days ºC). Thirty fish of each strain were collected for testing with PTD. These fish were processed for whole- head analysis as described previously.
30
Proc GLM in SAS system software was used to conduct tests in a general linear model framework for differences in growth and infection severity (dependent continuous variables) between strains (independent classification variable) for data collected during each sampling event. Wet weights over the course of the grow-out period were also compared using Proc GLM, in a simple regression analysis. This analysis used strain as an independent classification variable, days post-hatch as an independent continuous variable, and weight as a dependent continuous variable. Alpha was set at 0.05 for all tests of differences in growth and infection severity.
Poudre Rearing Unit - The Poudre Rearing Unit is located at an elevation of 2,347 meters above sea level in the Cache la Poudre Canyon, Northwest of Fort Collins, Colorado. The facility relies on surface water drawn from the Cache la Poudre River, which results in very slow growth at the facility during the winter months, when temperatures drop to near freezing from September through April (Figure 3.2). The facility has been positive for M. cerebralis since June of 1988. Fish produced at the Poudre Rearing Unit are typically brought to the facility in the late summer or fall of the year from the Bellvue Hatchery, near Laporte, Colorado, as 8 to 16-cm fingerlings. Fish reared at the facility are used as a catchable product or as replacement brood fish.
GR and Bellaire strain rainbow trout were brought to the Poudre Rearing Unit in late July, 2005, as 15-cm (43-45 g) M. cerebralis-negative fingerlings. Each lot consisted of 1,550 fish. The GR rainbow trout were seven months post-hatch, and the Bellaire rainbow trout were nine and a half months post-hatch. The age difference was necessary to match the sizes of the fish, due to the rapid early growth of the GR trout. Increase in size and age at exposure has been demonstrated to reduce infection severity in rainbow trout (Markiw 1992, Ryce et al. 2005). The GR rainbow trout were younger and therefore presumably more susceptible to infection as a function of age than the Bellaire rainbow trout when brought to the facility. The adipose fin was removed from GR rainbow trout two weeks prior to transport to ensure the fish could be easily identified when samples were collected.
The fish were held together in a single (1.8 m x 30.4 m) raceway, with a flow of
3,218 to 3,407 liters per minute. Fish were fed ad libitum with demand feeders during the summer months, and a daily maintenance ration 0.5%-2% body weight during the winter months when temperatures were below 2º C. Growth was monitored for one year at the facility.
Samples were collected for histological examinations and myxospore counts four
months (970 degree-days ºC) after the fish were transported to the facility. Thirty fish of the GR and Bellaire rainbow trout were collected for the evaluations. The fish were euthanized with tricaine methanesulfonate, then weighed and measured. Heads were removed from the fish and split in two equal halves along the dorsal midline for histological analysis and pepsin-trypsin digest (PTD).
Subsequent samples of 30 fish of each strain were collected at eight months
(1,039 degree-days ºC) and one year (1,617 degree-days ºC) after the fish were brought to
31
the facility. The entire head of each fish was collected and processed with PTD as described above in these samples. Average myxospore counts were compared between the two strains for each of the three sampling events. As with the data collected from the Chalk Cliffs evaluation, Proc GLM in SAS System software was used to test for differences in growth and infection severity for data collected during each sampling event, and alpha was set at 0.05 for all tests.
RESULTS
Chalk Cliffs Rearing Unit Growth – At the Chalk Cliffs Rearing Unit, growth as measured by average weight in the GR strain was much faster than the Tasmanian strain (Figure 3.3). A simple linear regression model with days post-hatch as an independent continuous variable and strain as an independent classification variable resulted in a very good fit (R2 = 0.8914). Both strain (F [1, 19] =23.70, P < 0.0001) and days post-hatch (F [1, 19] =132.23, P < 0.0001) were found to be significant parameters in this model. More complicated models were explored, with similar results. Growth differences were also quite different when direct length measurements were compared. At nine and a half months post-hatch, average length of GR strain was 23.6 cm (n = 60, SD = 1.5), and 18.5 cm (n = 60, SD = 2.4) for the Tasmanian strain. At one year post-hatch, the GR strain averaged 28.4 cm (n = 50, SD = 2.8), while the Tasmanian strain averaged 22.3 cm (n = 50, SD = 3.3). These differences were significant between the two strains during both the first (F [1, 118] =199.26, P < 0.0001) and second (F [1, 98] = 100.85, P < 0.0001) sampling events. M. cerebralis Infection. – Statistical test results for comparison of the infection severity and prevalence in the two strains for all of the sampling events at the Chalk Cliffs Rearing Unit are provided in Table 3.1. Samples collected at three months post-hatch were identified as negative with histology in both the GR and Tasmanian rainbow trout. Samples collected at five months post-hatch also resulted in negative results for both histology and PTD in both strains. At nine and a half months post-hatch, infection prevalence in the GR strain was 73.3%, and prevalence in the Tasmanian strain was 96.7% Average whole-head myxospore count in the GR strain was 5,175 (n = 30, SD = 7,643), compared with 48,883 (n = 30, SD = 50,825) in the Tasmanian strain. The differences in myxospore counts were highly significant (F [1, 58] = 21.70 P < 0.0001). Poudre Rearing Unit Growth - At the Poudre Rearing Unit, size in the GR rainbow trout was closely matched to the Bellaire rainbow trout for the first four months at the facility, with the GR rainbow trout averaging 24.3 cm (n = 30, SD = 2.1) versus 23.6 cm (n = 30, SD = 2.2) for the Bellaire rainbow trout. These differences were not significant (F [1, 58] = 1.86, P = 0.1779). At eight months, growth in the GR strain was slightly better (26.1 cm, n = 30, SD = 17.2) than the Bellaire (24.9 cm, n = 30, SD = 4.0), but the difference was not
32
significant (F [1, 58] = 2.31, P = 0.1340). By one year on the facility, the GR rainbow trout (35.0 cm, n = 30, SD = 4.3) were significantly (F [1, 58] = 19.07, P < 0.0001) larger than the Bellaire strain (30.4 cm, n = 30, SD = 3.7).
Average weights of the two strains followed the same pattern as the lengths. After four months on the facility, the GR rainbow trout averaged 173.0 g (n = 30, SD = 48.0), and the Bellaire rainbow trout averaged 171.0 g (n = 30, SD = 44.5). These differences were not significant (F [1, 58] = 0.04, P = 0.8373). After eight months on the on the facility, the GR strain averaged 190.5 g (n = 30, SD = 40.1), while the Bellaire strain averaged 180.1 g (n = 30, SD = 60.5). Again, the weights were not significantly different (F [1, 58] = 0.61, P = 0.4370). When sampled at one year on the facility, the GR strain averaged 493.1 g (n = 30, SD = 132.9 g), and the Bellaire strain averaged 375.4 g (n = 30, SD = 122.1). Despite being an equivalent size, but younger than the Bellaire rainbow trout at the beginning of the grow-out period, the GR strain were significantly heavier (F [1, 58] = 12.74, P = 0.0007) than the Bellaire strain.
M. cerebralis Infection - Testing for M. cerebralis resulted in identification of significantly higher prevalence and infection severity in the Bellaire rainbow trout than in the GR strain at the Poudre Rearing Unit (Figure 3.4). After four months, no infection was found in the head cartilage of any of the GR rainbow trout with histology, while lesions were found in 43.3% of the Bellaire rainbow trout. Histological scores in the Bellaire rainbow trout were low, with an average of 0.57 (SD = 0.73) on the McConnell- Baldwin scale of 0 – 5. No myxospores were found in any of the GR rainbow trout (n = 30) after four months. Prevalence of infection in the Bellaire strain as measured by PTD was 46.7% (n = 30), with an average (half-head) myxospores count of 3,657 (SD = 7,044).
Samples collected for PTD analysis after eight months resulted in only three of the GR rainbow trout identified as infected, with an average whole-head myxospore count of 3,440 (n =30, SD = 20,445). All of the Bellaire rainbow trout were found to be infected, with an average whole-head myxospore count of 84,993 (n = 30, SD = 86,791).
Samples collected after the two strains had been reared for one year on the facility
identified none of the GR rainbow trout (n = 30) as infected, while 90% (n = 30) of the Bellaire rainbow trout were identified as infected, with an average myxospore count of 361,099 (SD = 376,794 ) per fish.
Statistical test results for comparison of the infection severity and prevalence i

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